The Ubiquitin Proteasome System regulates essentially every cellular process and its misregulation can cause or potentiate disease. The 20S core proteasome is the endpoint of this process and its internal active sites degrade proteins to small peptides. However, there is a gap in our knowledge about how ubiquitinated proteins are recognized and then injected into the 20S for degradation. Two different ATP-dependent complexes have been shown to catalyze the degradation of ubiquitinated proteins, the 19S regulatory particle and P97 (VCP/CDC48). The 19S associates with the 20S to make the 26S proteasome; however, we do not understand how the 19S's molecular machinery-its ring of ATPases-uses ATP to bind and translocate substrates into the 20S. Even less is understood about how P97 catalyzes protein degradation including whether or not it even associates with the proteasome. Our long-term goal is to understand how protein degradation is regulated and to develop modulators that specifically target these regulatory mechanisms, which can be used as research tools or therapeutic agents. The overall objective of this application, which is the next step toward attaining this long- term goal, is to elucidate how the proteasomal ATPases and P97 function at a molecular level to facilitate protein degradation. The rationale for this objective is that detailed molecular models of how ubiquitinated proteins are processed for degradation are needed to understand how their misregulation is involved in disease. The objective of the application will be attained by pursuing two specific aims. The first aim will determine how allosteric regulation in the proteasomal ATPases control the position and timing of ATP hydrolysis to properly coordinate substrate degradation. Various biochemical and biophysical approaches will be taken utilizing both archaeal and eukaryotic model systems supported by yeast genetics. The second aim will determine how P97 catalyzes substrate degradation by the proteasome. Similar enzymological approaches will be taken using in vitro reconstituted systems, and the importance of a putative P97-20S interaction will be evaluated in mammalian and yeast model systems. This approach is innovative because we have generated a novel experimental system that will allow us to investigate the specific roles and functions of these enzymes and apply these findings to cell models in novel ways to determine their functional and biological roles. These outcomes are expected to have an important positive impact because they identify regulatory features of the UPS that have been missing from our under- standing of ubiquitin-dependent protein degradation. This contribution is significant because an understanding of how these molecular machines catalyze protein degradation is essential for understanding how this critical process can be misregulated in diseases such as cancer, neurodegenerative disease and aging. Such insights will lay the foundation for the development of new therapeutic strategies to specifically inhibit or activate these separate degradation pathways.